Abstract

Dicyclopentadiene (DCPD) is considered a pollutant due to its strong, persistent odor. Thirty five bacterial species were isolated from soil contaminated with C5+, which consists of low molecular weight hydrocarbons such as benzene, toluene, styrene, cyclopentadiene and dicyclopentadiene. All of these species were identified by partial 16S rRNA gene sequencing. The isolated genomic DNAs from these bacteria were spotted on a master filter in denatured form. These filters were hybridized with total community DNAs isolated from soil exposed to DCPD in order to determine the effect of DCPD on the soil microbial community, by comparison with the results obtained for an untreated control. Incubation of soil with DCPD enriched a Sphingomonas sp., while incubation with DCPD in the absence of soil gave enrichment of a Pseudomonas sp. Significant formation of oxygenated DCPD derivatives was observed if the Pseudomonas sp. was inoculated into a medium containing sterilized soil, minimal salts and DCPD. The results indicate that bacterial isolates with DCPD degrading potential can be identified with RSGP and can be isolated from C5+ contaminated sites.

Introduction

Ethylene production by pyrolysis, scheduled to rise in Alberta to 5x109 kg per year, yields hydrocarbon by-products (collectively referred to as C5+) that include BTEX (Benzene, Toluene, Ethylbenzene and Xylene), cyclopentadiene, dicyclopentadiene (DCPD) and others. Pyrolysis plants contain soils and sludges with significant C5+ contamination. There is a strong interest in assessing whether this contamination can be removed by bioremediation. Research is currently focused on (i) DCPD removal, and (ii) intrinsic bioremediation of C5+ components. Industrial interest in DCPD removal stems from its recalcitrance and pungent smell. The detection limit for the human nose is 5.7 ppb1. Bioremediation field trials have indicated that, after nutrient addition and auguring to provide air access, the BTEX components are removed relatively rapidly, leaving DCPD as the major contaminant. Laboratory studies by Stehmeier et al.2 indicated that environmental microbial consortia are able to mineralize DCPD to some extent and can form oxygenated derivatives. The objective of this paper is to indicate the organisms in the consortium responsible for DCPD transformation. This is done by tracking a large number of bacteria present at a contaminated site simultaneously by reverse sample genome probing (RSGP). For RSGP analysis3–5 a set of genomically distinct bacteria is isolated from the target environment and the denatured genomes are spotted on a "genome chip" or master filter (Fig. 1A: 1 to12) together with different amounts of DNA from an internal standard (Fig. 1A: i1 to i4). Once these filters have been made, analysis involves: (i) extraction of total DNA from incoming samples, (ii) random labeling of sample DNA (containing different chromosomal DNAs at a weight fraction Fx each) spiked with the internal standard, (iii) hybridization of the resulting probe with a master filter (Fig. 1B), and (iv) analysis of hybridization intensities for individual dots from an image on film or a phospho-imaging plate to calculate the fractions (the fx values) for community members represented on the filter (Fig. 1C).

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